Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype.     

A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States

Several quantitative trait loci (QTLs) that influence developmental dyslexia (reading disability [RD]) have been mapped to chromosome regions by linkage analysis. The most consistently replicated area of linkage is on chromosome 6p23-21.3. We used association analysis in 223 siblings from the United Kingdom to identify an underlying QTL on 6p22.2. Our association study implicates a 77-kb region spanning the gene TTRAP and the first four exons of the neighboring uncharacterized gene KIAA0319. The region of association is also directly upstream of a third gene, THEM2. We found evidence of these associations in a second sample of siblings from the United Kingdom, as well as in an independent sample of twin-based sibships from Colorado. One main RD risk haplotype that has a frequency of approximately 12% was found in both the U.K. and U.S. samples. The haplotype is not distinguished by any protein-coding polymorphisms, and, therefore, the functional variation may relate to gene expression. The QTL influences a broad range of reading-related cognitive abilities but has no significant impact on general cognitive performance in these samples. In addition, the QTL effect may be largely limited to the severe range of reading disability.     

Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia typeII

Tyrosinemia typeII (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG→TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia typeII. Received: 8 October 1997 / Accepted: 29 October 1997     

Mutations associated with variant phenotypes in ataxia-telangiectasia.

We have identified 14 families with ataxia-telangiectasia (A-T) in which mutation of the ATM gene is associated with a less severe clinical and cellular phenotype (approximately 10%-15% of A-T families identified in the United Kingdom). In 10 of these families, all the homozygotes have a 137-bp insertion in their cDNA caused by a point mutation in a sequence resembling a splice-donor site. The second A-T allele has a different mutation in each patient. We show that the less severe phenotype in these patients is caused by some degree of normal splicing, which occurs as an alternative product from the insertion-containing allele. The level of the 137-bp PCR product containing the insertion was lowest in two patients who showed a later onset of cerebellar ataxia. A further four families who do not have this insertion have been identified. Mutations detected in two of four of these are missense mutations, normally rare in A-T patients. The demonstration of mutations giving rise to a slightly milder phenotype in A-T raises the interesting question of what range of phenotypes might occur in individuals in whom both mutations are milder. One possibility might be that individuals who are compound heterozygotes for ATM mutations are more common than we realize.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

Identification of Flagellar Motility Genes in Yersinia ruckeri by Transposon Mutagenesis

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersion-applied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model.     

Application of Fluorescent Amplified Fragment Length Polymorphism for Comparison of Human and Animal Isolates of Yersinia enterocolitica

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype.     

A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1-16q12.2

Familial amyotrophic lateral sclerosis (FALS) affects 5%-10% of cases of amyotrophic lateral sclerosis (ALS) and is inherited as an autosomal dominant condition with incomplete penetrance. One-fifth of these cases of FALS are associated with mutations in copper/zinc-dependent superoxide dismutase (SOD1), but the gene defect in the remaining 80% of familial cases is, as yet, unknown. We have carried out a preliminary genome screen, using a U.K. resource of families lacking SOD1 mutations, to identify other potential disease loci and have identified a putative locus on chromosome 16q12.1-q12.2. The region associated with disease was further refined in the major family that contributed to this result and was localized to D16S409-D16S3032, a 14.74-cM genetic interval that corresponds to a physical distance of 6.6 Mb, which coincides with a region independently identified by two further research groups in the United States and the United Kingdom.     

Molecular epidemiology and phylogeny reveal complex spatial dynamics in areas where canine parvovirus is endemic

Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described.     

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains

Aims: To establish PCR‐based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype‐causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. Methods and Results: Novel assays were developed relying on detection of mutant allele‐specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. Conclusions: The described methods were specific, rapid and simple to perform and interpret. Significance and Impact of the Study: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.     

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.     

Association of the human Lewis blood group Le(a-b-c-d-) with the failure of expression of alpha-3-L fucosyltransferase

Two unrelated individuals are reported who lack alpha-3-L-fucosyltransferase activity in their serum and saliva. Both were blacks, one from the United States and the other from South Africa. No other of the tested members of their families lacked this enzyme. A survey of more than 2000 serum samples from both black and white South African blood donors, black United States donors and white United Kingdom donors failed to disclose another example of a serum deficient in alpha-3-L-fucosyltransferase activity. The two individuals lacking in alpha-3-L-fucosyltransferase activity both had the Lewis blood group phenotype Le(a-b-c-d-). No other persons with this phenotype have been reported. The absence of Lec activity in the two individuals who are deficient in alpha-3-L-fucosyltransferase is consistent with the interpretation that alpha-3-linked L-fucose is an essential part of the antigenic determinant recognised by the anti-Lec reagent used in this investigation.     

Russian Contribution to OPV

Abstract. Field trials and the first mass immunization campaign using Oral Poliomyelitis Vaccine (OPV) from the strains derived by Albert Sabin were carried out in the former U.S.S.R. in 1959 under the guidance of Mikhail Chumakov. The results obtained during these trials contributed greatly to the general acceptance of OPV as a safe and efficient preparation. Mechanisms responsible for the attenuated phenotype of the Sabin strains have long been studied in Russia. For the first time, recombination analysis was used for mapping genetic determinants of attenuation, which were found to reside largely in the 5′ half of the Sabin strains RNA. It has been established that the phenotype effect of a subset of the major attenuating mutations is due to the impairment of the translation initiation.     

Comparison of Cryptosporidium parvum Viability and Infectivity Assays following Ozone Treatment of Oocysts

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4°C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4′,6′-diamidino-2-phenylindole–propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a “gold standard” until suitable alternative viability surrogates are identified for disinfection studies.     

Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.     

Mutational analysis of the starch utilization system in the yeast Saccharomyces cerevisiae

Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed. For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed. The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes. As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid. Such reversion has also been observed for the transformed stall mutant. Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment. All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.     

Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.     

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.